In India, mutations in Cytochrome P450 (CYP1B1) are a predominant cause of not only primary congenital glaucoma (PCG) but also involved in primary open angle glaucoma (POAG) and juvenile onset glaucoma (JOAG). After ethical clearance, 100 POAG patients, 30 primary angle closure glaucoma (PACG) patients and 130 ethnically matched controls were recruited in this study. Genomic DNA was isolated from the blood and screened for p.Arg368His mutation in CYP1B1 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). On PCR-RFLP, 10/100 cases (10%) were found positive for Arg368His mutation. In North Indian POAG cases studied, p.Arg368His mutation was found only in heterozygous state. The frequency of p.Arg368His CYP1B1 mutation in heterozygote state (10.0%) observed in our study in North Indian POAG patients is the highest in comparison to frequency observed in other ethnic groups from Southern and Eastern India.